Evaluation of Different Mycobacterial Species for Drug Discovery and Characterisation of Novel Inhibitors of Mycobacterium Tuberculosis

Abstract

<p>Tuberculosis (Tb) has plagued mankind for many centuries and is still a leading cause of death worldwide. A worrying development is the emergence of drug-resistant Tb that poses further challenges to the control of the disease. The global Tb burden and high mortality rate indicate that new drugs are needed for Tb treatment. While no new anti-Tb agents have been introduced to the market for about three decades, drugs with novel mechanisms of action can amend the current Tb treatment regimen and may provide an effective solution to drug resistance. The main objectives of this study were to identify an appropriate in vitro model that could be used for anti-Tb drug high-throughput screening (HTS), and to use this model to identify a novel candidate anti-tubercular drug and its cognate cellular target. A sensitive growth inhibition assay was set up with a GFP-labelled Tb vaccine strain, M. bovis BCG, using standard first and second line anti-tubercular drugs. HTS of the drug libraries was performed with various in vitro models to evaluate their efficacy for use in anti-Tb drug discovery. Approximately 50% of the M. tuberculosis inhibitors were not detected in screening with the surrogate species, M. smegmatis; whereas, only 21% of hits were not detected with M. bovis BCG. A comparative genomic study revealed that 97% of M. bovis BCG proteins, compared to 70% in M. smegmatis have conserved orthologues in M. tuberculosis H37Rv. Therefore, M. bovis BCG represented a more sensitive model than M. smegmatis for detecting anti-M. tuberculosis compounds. M. bovis BCG was then used to screen for novel anti-Tb agents by HTS of compound libraries and various plant extracts, followed by validation of new compounds in M. tuberculosis H37Ra. A number of novel M. tuberculosis inhibitors were identified, including sappanone A dimethyl ether from plant sources and compounds NSC112200 and NSC402959 from NIH chemical libraries. The inhibitors that were validated using M. tuberculosis H37Ra were also validated in the virulent Tb strain, M. tuberculosis H37Rv. In addition, their activity was further investigated using a suite of other clinically important human pathogens. One of the key anti-mycobacterial hits identified in this study, NSC402959, has previously been detected in screens for compounds that inhibit ribonuclease H (RNase H), an enzyme that is required for a number of essential cellular processes. NSC402959 inhibited RNase H proteins from HIV as well as from E. coli. Since HIV and Tb are major pandemics, previously-known anti-HIV RNase H compounds were imported and tested for their anti-proliferative activity towards M. tuberculosis H37Ra. HIV RNase H inhibitors, NSC35676, NSC112200, NSC133457 and NSC668394, exhibited good anti-mycobacterial activity in this study. In silico analysis suggested a plausible interaction of these inhibitors with mycobacterial RNase HI. A biochemical assay further confirmed NSC112200 to be specific against RNase HI from M. tuberculosis. These interesting inhibitors were not only structurally different from existing anti-Tb drugs but some of them were also non-toxic to mammalian cells and may have a unique mechanism of action. Thus, these compounds showed good potential for development as dual inhibitors of Tb and HIV; therefore, future studies in animal infection models to determine their dual anti-mycobacterial and anti-HIV activities are warranted.</p>